FORMULATION AND EVALUATION OF KETOCONAZOLE MICROEMULGEL WITH MIXTURE OF PENETRATION ENHANCERS

Abstract

The present research work was to develop ketoconazole loaded microemulgel formulation. The main objective was to enhance the penetration capability of ketoconazole by incorporating penetration boosters and to administer the drug in a sustained release fashion. Currently available dermal creams refuse to provide intended action as needed. Screening of oils, surfactants, and co-surfactants was done by the construction of pseudo ternary phase diagrams with 2% ketoconazole. Compatibility studies like FT-IR, DSC were performed to determine incompatibilities. The microemulsion was characterized for droplet size, zeta potential, viscosity thermodynamic stability. Moreover, for enhancement of patient compliance the optimized microemulsion was modified into microemulsion based gel. Exvivo studies were carried out for microemulgel using Franz diffusion cell by help of porcine skin membrane. The antifungal activity of microemulgel was evaluated using cup plate method incorporating Candida albicans (MTCC Code: 3018). The optimized microemulsion had a composition of 20% Oleic acid: coconut oil (2: 1), 34.06% Tween 80: Propylene glycol (2: 1), and 43.94% water and was later incorporated into polymeric gel base. The microemulgel exhibited IOhr sustained release profile when compared to the Kz cream@. Invivo investigation i.e; skin irritation test on albino mice was done by grouping into standard [Kz cream@], control[placebo], test[microemulgel] and it was identified that no irritation caused by microemulgel as well as standard Kz cream. The control showed signs of irritation as it does not possess active moiety. The optimized microemulsion showed 99.02% drug loading and 98.07% transmittance.The thermodynamic stability, sustained drug release with greater penetration and enhanced activity due to the presence of oleic acid in microemulgel warrant its application as an excellent formulation for treating opportunistic fungal infections.

Specification

Description:FIELD OF INVENTION:
[0001] The main content of this research is to Formulation and Evaluation of Ketoconazole Microemulgel with Mixture of Penetration Enhancers.

BACKGROUND:
[0002] The present research work was to develop ketoconazole loaded microemulgel formulation. The main objective was to enhance the penetration capability of ketoconazole by incorporating penetration boosters and to administer the drug in a sustained release fashion. Currently available dermal creams refuse to provide intended action as needed. Screening of oils, surfactants, and co-surfactants was done by the construction of pseudo ternary phase diagrams with 2% ketoconazole. Compatibility studies like FT-IR, DSC were performed to detem1ine incompatibilities.

[0003] The present research work was to develop ketoconazole loaded microemulgel formulation. The main objective was to enhance the penetration capability of ketoconazole by incorporating penetration boosters and to administer the drug in a sustained release fashion. Currently available dermal creams refuse to provide intended action as needed. Screening of oils, surfactants, and co-surfactants was done by the construction of pseudo ternary phase diagrams with 2% ketoconazole. Compatibility studies like FT-IR, DSC were performed to detem1ine incompatibilities.

[0004] In vivo investigation i.e.; skin irritation test on albino mice was done by grouping into standard [Kz cream(r)], control[placebo], test[microemulgel] and it was identified that no irritation caused by microemulgel as well as standard Kz cream. The control showed signs of irritation as it does not possess active moiety. The optimized microemulsion showed 99.02%drug loading and 98.07% transmittance. The thermodynamic stability, sustained drug release with greater penetration and enhanced activity.

SUMMARY:
[0005] Topical formulations are utilised for localised effects at the point of application due to medication penetration into the underlying layers of skin or mucous membranes. It allows the use of medications with a short biological half-life and a narrow therapeutic window to extend the duration of activity.1.2 The natural barrier for topical distribution is skin3 4 which makes drug delivery problematic. Taking this into account, microemulsions are formed that have low skin irritation a high drug loading capacity, and may minimise the diffusion barrier of the Stratum corneum and boosting drug absorption.
[0006] Oleic acid, (9Z)-Octadec-9-enoic acid classified under monounsaturated omega-9 fatty acid acts as penetration booster by disruption of skin s barrier functionality and thereby enhancing drug partitioning into Stratum Corneum and it boosts the delivery of both lipophilic and hydrophilic drugs. Due to the penetration enhancer property oleic acid possess wide range of applications in pharmaceutical formulations like excipient and in the solution phase synthesis of the nanoparticles.

[0007] Microemulsions are thermodynamically stable isotopically transparent dispersions of two immiscible liquids such as oil and water stabilised by an interfacial layer of surfactant molecules with a size range of I0-200nrn and very low interfacial tension.8 It consists mostly of oil, surfactant (SA) and water with varying amounts of cosurfactant (Co-SA). This combination is clear and steady.9 The current study focuses on ketoconazole microemulsion based gel with penetration enhancers for the treatment of topical infections.

[0008] Ketoconazole,2,2,2-trideuterio-1-[4-[4-[[(2R,4S)-2-(2,4-dichlorophenyl)-2-(imidazol- l- y methyl)-1,3-dioxolan-4-yl] methoxy] phenyl] piperazin-1-yl] ethenone imidazole antifungal drug. preferentially inhibits ergosterol production in fungal cell wall as a result occurrence of flaws in fungal cell wall and there by acting as a fungistatic agent.

[0009] The optimized formulation comprising 2% ketoconazole, 20% oil mix (Oleic acid: coconut oil:2:1), 34.06% Smix (Tween 80: PG:2:1) and 43.94 % water was selected on the basis of drug loading, percent transmittance, thermodynamic stability and ex- vivo drug release. The chosen micro emulsion showed 99.02% drug loading and 98.07% transmittance. The microemulsion when loaded into different grades of Carbopol gel colour change occurs indicating chemical interaction. The microemulsion was loaded into a gel base comprising of 1% HPMC K4M and 2% sodium alginate in the ratio of 1:1. The microemulgel containing 1% HPMC K4M and 2% sodium alginate in the ratio of 1:1 was white in appearance, and smooth in consistency. It did not exhibit any colour change. The ex- vivo study was done on porcine skin due to unavailability of human cadaver nail plate. The microemulgel showed a slow release of •19.82% whereas Kz cream@ showed a release of 48.47% at the end of 10th hr. The microemulgel was stable for a period of two months. In-vivo study revealed no irritation on animal skin. Thus, ketoconazole and oleic acid microemulgel formulation could show promising and beneficial results in treating opportunistic fungal infections.

DETAILED DESCRIPTION OF FIGURES AND TABLES:
[0010] Table- I: Solubility profile of drug
[0011] Table-2: Construction of calibration curve of ketoconazole: Amax: 292.4nm

[0012] Figure 1: Calibration curve of ketoconazole in pH 5.5: methanol (I: 1) ratio.

[0013] Figure-2: (a) FT-IR Spectrum of pure drug (b) FT-IR Spectrum of microemulgel

[0014] Figure-3: (a) DSC of pure drug (b) DSC of microemulgel

[0015] Table-3: Formulation batches of Ketoconazole Microemulsions.

[0016] Figure-4(a): Ternary plot of oil mix(l:1) +Smix (1:1) 4(b): Ternary plot of oil mix (2:1) +Smix (2:1)

[0017] Table 4: Measurement of Viscosity, pH, Mean droplet size, PDI, zeta potential and drug content

[0018] Figure-5: Measurement of particle size of microemulsion

[0019] Figure-6: Ex vivo comparative release study between rnicroemulgel and Kz cream(r)

[0020] Figure-7: Zone of inhibition of placebo and rnicroemulgels.

DETAILED DESCRIPTION:
[0021] Ketoconazole (KTZ) drug was a gift sample from Chalapathi Institute of Pharmaceutical Sciences, Guntur, India. Coconut oil (oil phase) was purchased from a local vendor in Guntur (India). Oleic acid (oil phase) and Tween 80 (surfactant) from Loba Chemie Pvt.Ltd and propylene glycol (co-surfactant) from Qualigens. Double distilled water was incorporated aU over the formulation procedure and all other chemicals were of analytical grade. Ketoconazole solubility was tested using a variety of oils, surfactants and cosurfactants. Solubility investigations were carried out by using an addition of excessive dose of the drug to 2mL of the vehicle contained in a 5mL glass vial. The drug was dissolved by heating the mixture in a water bath at 50 C with vertexing, then mixing for 2hr in an orbital shaker at 25 0C ±10 C (Rerni). Heat was used to create the kinetic energy needed to disrupt the intermolecular interactions that hold the solute molecules together.

[0022]The supernatant of an equilibrated combination centrifuged at 5000rpm for 15rnin was examined using a UV-Vis spectrophotometer at 292.4nm Construction of standard plot of ketoconazole Accurately weighed quantity of drug (10mg) was solubilised in appropriate quantity of methanol and diluted to J0mL with saline phosphate buffer of pH 5.5 and ethanol in the ratio of 1: I to obtain I 000 g/ml solution and from above solution 1mL was pipetted out and made up to 10mL with buffer to get 100 g/ml solution. Serial dilutions were made from above solution and analysed spectrophotometrically at an Amax of 292.4nm reach equilibrium for l 5min.

[0023] All ketoconazole containing microemulsions were then kept at room temperature and monitored. Following a 24hr interval for phase separation. Characterization of microemulsion the optimised microemulsion was evaluated for droplet size, zeta potential (nanoparticle analyser SZ-100, Horiba scientific), pH (LABINDIA), transmittance, percent drug loading, viscosity (Brookfield viscometer), thermodynamic stability against centrifugation at 5000 rpm for l 5min.

[0024] Droplet size determination: The Dynamic light scattering particle size analyser (nanoparticle analyser SZ-100, Horiba scientific) was used to determine the droplet size of microemulsion. Dilution was performed by taking 1mL of microemulsion and diluting it to I 0mL with water before obtaining measurements.

[0025] PH: The pH electrode f pH meter was calibrated with acetate buffer of pH 5.5 and a 1% aqueous formulation solution before being submerged until the variability in readings ended and a consistent reading was achieved .

[0026] Viscosity: Approximately 1g of microemulsion was poured on the plate of the Brookfield viscometer and allowed to settle for 5-10min before the spindle(S62) was placed on the plate containing micro emulsion and rotated at 5rpm. The temperature was kept at 25 C and the appropriate dial reading was recorded in cps.

[0027] Zeta potential: Zeta potential is a process of scattering electrophoretic light and it was measured using HORIBA SZ-100 analyser by keeping the sample in cuvette having cathode and anode with the help of syringe.

[0028] Thermodynamic stability: Microemulsion stability was tested by centrifuging it for 15min at 5000rpm. Any changes such as phase separation and clarity were visually observed. As the centrifugation technique produced the intend d outcomes no freeze-thaw cycles or heating and cooling cycles were performed.

[0029] Drug content: The drug content of the microemulsion corresponding to 200mg was determined by double beam UV-Visible spectrophotometer after dilution with methanol at 292.4 run against methanol as blank. The total drug content was estimated by using the formula
%Drug content = (actual drug content / theoretical drug content) x 100

[0030] Formulation of microemulsion loaded microemulgel: Gelling agents were applied to these systems to improve the microemulsion viscosity and thereby increasing its retention period. Gelling agents Like HPMC K4M and sodium alginate were soaked overnight to obtain consistent rnicroemulgels. The optimum ketoconazole loaded microemulsion was introduced into the microemulgel base.

[0031] Characterization of microemulsion loaded microemulgel: The pH (LABINDIA), viscosity (Brookfield viscometer) and drug content of the optimized microemulgel were measured.

[0032] Drug content: The drug content was determined by dissolving lg of microemulgel in l00mL of pH 5.5 phosphate buffer: ethanol (I: 1) buffer. 1mL of this was taken and diluted up to 10mL with phosphate buffer. Because ketoconazole was insoluble in water it was dissolved in phosphate buffer, which had the maximum solubility. This solution was sonicated for 30min and then filtered. There was no drug precipitate seen since it disintegrated entirely following sonication. the absorbance was measured spectrophotometrically at Amax of 292.4nm.

[0033] Ex vivo skin penetration studies: The experiments were carried out on excised porcine skin utilising Franz diffusion cell construction. Phosphate buffered saline (PBS) pH 5.5 and ethanol (1:1) was employed as receptor medium and cell contents were kept at 37.0 C. For lipophilic drugs such as ketoconazole, solubility would be a rate limiting step via skin absorption in receptor fluid potentially affecting overall flow. As a result, methanol was used as solubilizer for ketoconazole without affecting skin integrity. I mL aliquot was removed from the receptor chamber at appropriate time intervals and replaced with new buffer.

[0034] Each experiment was carried in triplicates. The residual formulation was collected from the skin and weighed at end of ex vivo drug release testing. The pig skin was made to small pieces and put in 10mL of phosphate buffer pH 5.5 and methanol (1:1) in water bath for 30min followed by l 5min sonication and centrifugation at 5000rpm. After diluting I mL of sample with phosphate buffer pH 5.5: methanol (1:1). The quantity retained was measured spectrophotometrically at 292.4 nm.

[0035] Antifungal activity: By employing the agar plate diffusion method the microemulsion loaded gel24 and placebo formulation was evaluated for activity on fungal strain Candida albicans which was cultured on dextrose agar medium. A suspension of Calbi cans of I 00 1 was inoculated in three agar plates now plates were bored and filled with 0.3g of formulation and control, after filling the plates were incubated at 37 C for a period of 24hr and measured the zone of inhibition. Experiment performed in triplicates.

[0036] In-vivo study: All the experimentation procedures were reviewed and approved by IAEC (Approval number: I 6/IAEC/CLPT/2021-22). The institution of reviewers committee was Acharya Nagarjuna University, Guntur.

[0037] Accelerated Stability studies: The optimized gel formulation was taken in collapsible aluminium tubes and stored at 5 3 C, 25 2 C /60% 5%RH and 40 2 C/75% 5%RH for 3months. Samples were analysed for appearance, drug content, release profiles.

[0038] Solubility studies: From the solubility studies, it was evident that ketoconazole was highly soluble in Coconut oil and nigella oil mixture among the different oils and then oleic acid+coconut oil. Drug showed maximum solubilization in tween 80+propylene glycol among three combinations followed by tween 20+PEG400.

[0039] Compatibility studies FT-IR Study: The infrared spectrum of ketoconazole pure drug in figure-2(a) shows strong absorption bands at 1643.57cm"1(C=O carbonyl stretching) and IQ24.52cm"1<C-O aliphatic ether stretching), l 242.78cm 1(C-O cyclic ether stretching). For microemulgel in figure-2(b) the absorption bands were observed at l 64l. l 7cm 1 and at 1084.23cm 1 respectively indicating no interactions.

[0040] DSC Studies: In DSC study, a sharp endothermic peak was appeared at 151.91 c denoting the melting point of ketoconazole drug and the peak in microemulgel was similar to that of pure drug denoting no possible interactions.

[0041] Formulation of microemulgel: The optimized formulation of microemulsion was incorporated into Carbopol gel base which was previously prepared then an immediate colour changes to pink was observed due to interactions of oil and drug. Carbopol was not preferred.27 So, HPMCK.iM and sodium alginate in 1: l proportion yielded required consistency hence it was chosen for formation of microemulgel and the resultant microemulgel was evaluated for drug release. drug content and antifungal activity.

[0042] Characterization of microemulsion loaded microemulgel: The pH, viscosity, and drug content of microemulgel were found to be 6.12 0.15, 7510 cps. and 96.25%.

[0043] Ex vivo skin penetration studies: A comparison of conventionally marketed Kz cream and microemulgel loaded with ketoconazole microemulsion was conducted. Figure 6 shows a study of the ex vivo release of a microemulgel against the marketed formulation (Kz cream(r)). During the beginning hour, the drug release from microemulgel and Kz cream(r) were found to be 4.73 0.03% and 11.65 0.34, respectively. This demonstrated that the release of drug from Kz cream(r) was three times that of microemulgel formulation. At the last of tenth hour, 19.82 0.22% release was found out from microemulgel as well as 48.47 0.06% was released from conventional Kz cream(r).

[0044] For treatment of infections caused by fungus a formulation with sustained release is recommended as it decreases the frequency of application when in comparison to the conventional cream. The skin retention study states that there were no significant variations among the two formulations. The percent of microemulgel and Kz cream(r) remained on the porcine skin membrane was be 23.56% 0.63 and 21.08% 0.36 respectively.

[0045] The vivo study: Grouping of animals and IAEC approval albino mic w re grouped 24hr prior to experimentation into three namely standard control and test each group comprising of five mice. All the animals were habituated to 12hr dark cycle and 12hr light cycle. The study protocols were reviewed and approved by the lAEC (Approval number: l6/IAEC/CLPT/2021-22). AII the experimentation was carried out in animal house of Chalapathi institute of pharmaceutical sciences.

[0045] Procedure: Skin irritancy test: Arrange the mice in aseptic condition by cleaning of experimental area with 70% v/v alcohol. The albino mice of200g weight were taken and the hair of the mice were scraped off up to l- 2cm2 on previous day of experiment. After that application of Kz cream (Standard) to group-I and application of oleic acid containing placebo to group-2 and formulated microemulgel(test) to group-3. Allow the mice to withstand for more than 24hr then observe for any irritation or redness on the skin. , Claims:Claims:
I/We Claim:
1. A process of developing ketoconazole loaded microemulgel formulation, comprising:
a ketoconazole;
the ketoconazole of 10mg was dissolved in diluted methanol was heated in a water bath at 500C with vortexing;
whereby mixed for 2hr in an orbital shaker at 25 ± I OC;
the methanol of 10mL is diluted with saline phosphate buffer of pH 5.5 and ethanol in the ratio of 1:1 to obtain 1000gg/ml solution;
the solution of 1mL was pipetted out and made up to 10mL with buffer to get 100gg/ml solution;
the mixture of an equilibrated combination centrifuged at 5000rpm for 15min was examined using a UV-V spectrophotometer at 292.4nm to reach equilibrium for 15min; and
all ketoconazole containing microemulsions were then kept at room temperature and monitored.

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